The internal anal sphincter (IAS) is responsible for ~70% of anal resting tone, a role that is critical for maintaining continence. Mechanisms regulating contraction in the IAS differ significantly from other regions of the large intestine where mixing and the forward propulsion of luminal contents predominate. Studies of the IAS by others have focused on mechanisms that sensitize the myofilaments to intracellular Ca2+concentration ([Ca]i) but without the continued entry of calcium into cells through voltage dependent calcium channels (VDCC) there can be no tone in the IAS. Likewise, any process (such as neural activity) that reduces VDCC activity will reduce tone. Our findings during the past funding period have led to the hypothesis that slow waves (SW) play a fundamental role in maintaining tone in the IAS. In the next funding period we examine the role ICC and a second interstitial cell type referred to as PDGFRa+ cells in the generation of SW and in inhibitory neuromuscular transmission (NMT) in the IAS. We will use a number of newly developed transgenic mice in which specific classes of cells express various fluorescent tags or Ca2+ indicators or have key genes deactivated. Ca2+ imaging studies will identify sites of SW origin and discover which cells respond to inhibitory motor innervation. Specific cell populations will also be collected for molecular analyses and patch clamp studies. When possible we will also apply these results to parallel studies of IAS from Cynomolgus monkeys, a close genetic relative of humans. Dysfunction of the IAS leads to substantial social isolation, loss of employment, and institutionalization as well as costing the health care system in the USA billions of dollars annually. In spite of this large health care burden the IAS is the least studied region of the GI tract. This proposal will bring studies of the IAS into the modern era of cellular molecular probes transgenic technology and rigorous investigation of mechanism. Such information is likely to provide novel rationales for treating defecation disorders involving inappropriate IAS tone.